Spectral imaging for vertical sectioning

ABSTRACT

A method and apparatus for performing optical microscopy in one to three dimensions employs a spectral self-interference fluorescent microscopy technique that includes providing at least one fluorescent microscopy sample, at least one objective lens, and at least one reflecting surface. The fluorescent sample is disposed between the objective lens and the reflecting surface, the distance from the sample to the reflecting surface being several to several tens times an excitation wavelength. Excitation light causes the fluorescent sample to emit light, at least a portion of which is reflected by the reflecting surface. The objective lens collects the reflected light and the light emitted directly by the fluorescent sample. The direct and reflected light interfere causing spectral oscillations in the emission spectrum. The periodicity and the peak wavelengths of the emission spectrum are spectroscopically analyzed to determine the optical path length between the fluorescent sample and the reflecting surface.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Patent ApplicationNo. 60/256,574 filed Dec. 19, 2000 entitled SPECTRAL IMAGING FORVERTICAL SECTIONING.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Contract NumberDBI-9812377 awarded by the National Science Foundation, Contract Number99-35201-8435 awarded by the U.S. Department of Agriculture and ContractNumber JPL-1213572 awarded by DARPA. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to optical microscopy, and morespecifically to optical microscopy in one to three dimensions thatprovides increased resolution while enhancing the specificity of opticalmeasurements.

Techniques for performing optical microscopy are known that are capableof providing high resolution three-dimensional (3-D) imaging. Suchoptical microscopy techniques have a number of advantages overnon-optical microscopy techniques such as electron microscopy andscanned probe microscopy. For example, optical microscopy can be used toview living tissue samples in their natural state, thereby enabling thestudy of complex biological mechanisms. In contrast, electron microscopytypically requires microscopy samples to be dried and exposed to avacuum, which would normally kill living tissue samples. Not only canoptical microscopy be used to view living tissue samples, but it canalso be used to map the interior of such samples in multiple dimensions.Scanned probe microscopy, in comparison, can typically only be used tomap surfaces of living tissue samples and is therefore incapable ofproviding information about the sample's interior. Optical microscopycan also be used with fluorescent probe technology to allow cellularcomponents of living tissue samples to be identified and mapped withsome degree of specificity.

Conventional techniques for performing 3-D optical microscopy includeoptical sectioning microscopy, scanning confocal microscopy, two-photonmicroscopy, and 4Pi confocal microscopy. Optical sectioning microscopytechniques typically comprise acquiring a series of images of amicroscopy sample by successively moving sections of the sample througha focal plane. Each image includes in-focus information from the samplesections in the focal plane and out-of-focus information from theremaining sections of the sample. The image information is then analyzedby computer to reconstruct the 3-D structure of the sample. Suchcomputer analysis typically employs at least one computationalde-convolution algorithm and reference data describing the “blur” causedby a single point source of light. Scanning confocal microscopytechniques typically comprise focusing a laser beam onto a spot in amicroscopy sample and detecting light through a pinhole focused onto thesame spot in the sample as the laser. Next, the focal point is scannedin three dimensions through the sample. Finally, light intensity isdetected as a function of the spot position to obtain a 3-D image of thesample.

However, conventional optical microscopy techniques such as opticalsectioning microscopy and scanning confocal microscopy have drawbacks inthat the depth resolution (i.e., the resolution in the verticalZ-direction) is, in general, worse than the resolution in the transverse(i.e., the X-Y) plane. As a result, the range of biological mechanismsthat can be studied by these conventional optical microscopy techniquesis limited.

For example, in order to study cellular functions such as cell cycle,development, motility, adhesion, and DNA replication, it is oftennecessary to perform precise 3-D localization of proteins withinprokaryotic cells. This would normally require an optical microscopytechnique capable of providing depth resolution on the order of at leastten to tens of nano-meters. However, conventional optical microscopytechniques such as optical sectioning microscopy and scanning confocalmicroscopy have traditionally only been capable of providing depthresolution on the order of half a micron.

It would therefore be desirable to have a technique for performingmulti-dimensional optical microscopy that can be used, e.g., to study awide range of biological mechanisms and sub-cellular processes in realtime. Such an optical microscopy technique would provide resolution onthe order of at least tens of nano-meters in at least one dimension. Itwould also be desirable to have an optical microscopy technique that canbe used with fluorescent probe technology to enhance the specificity ofoptical measurements.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, a method and apparatus forperforming optical microscopy in one to three dimensions is disclosedthat can be used, e.g., to study a wide range of complex biologicalmechanisms. Benefits of the presently disclosed optical microscopytechnique are achieved by employing spectral self-interferencefluorescent microscopy to determine an optical path length between atleast one fluorescent microscopy sample and a reflecting surface,employing variable standing wave illumination to extend the capabilitiesof the spectral self-interference fluorescent microscopy technique toprovide vertical sectioning of an arbitrary distribution of fluorescentsamples, and employing rotating aperture interferometric nanoscopy toprovide such sectioning along a plurality of axes to generate imageinformation suitable for reconstructing the three-dimensional structureof the sample distribution.

In one embodiment, the spectral self-interference fluorescent microscopytechnique employed by the presently disclosed invention comprisesproviding at least one fluorescent microscopy sample, at least oneobjective lens, and a first reflecting surface. The fluorescent sampleis disposed between the objective lens and the first reflecting surface.The fluorescent sample emits light, at least a portion of which isreflected by the first reflecting surface. The objective lens collectsboth the reflected light and the light emitted directly by thefluorescent sample. The direct and reflected light undergo constructiveand destructive interference, thereby causing spectral oscillations or“fringes” in the emission spectrum. The difference between therespective optical path lengths of the direct and reflected light issuch that only a relatively small change in wavelength is needed totransition between the constructive and destructive interferencepatterns. The periodicity and the peak wavelengths of the emissionspectrum are then spectroscopically analyzed to determine the opticalpath length between the fluorescent sample and the first reflectingsurface.

The spectral self-interference fluorescent microscopy technique iscombined with variable standing wave illumination to allow the heightdetermination capability of spectral self-interference fluorescentmicroscopy to be applied to an arbitrary vertical distribution offluorescent samples. The variable standing wave illumination techniqueemployed by the presently disclosed invention comprises configuring thefirst reflecting surface to reflect emission wavelengths and betransparent at an excitation wavelength, and providing a movablewavelength-independent reflecting surface configured to reflect theexcitation wavelength. Excitation light is then provided by a lightsource and directed to the movable reflecting surface along a verticalaxis, and the direct excitation light and the light reflected by themovable surface interfere to form at least one standing wave aligned inthe direction of the vertical axis. Next, the movable reflecting surfaceis moved to translate the standing wave, thereby effectively scanningthe vertical distribution of fluorescent samples through the standingwave. Such scanning causes fluorophores to emit light within a pluralityof thin sections of the sample distribution orthogonal to the verticalaxis.

The rotating aperture interferometric nanoscopy technique employed bythe presently disclosed invention comprises directing the excitationlight to the movable reflecting surface along a plurality of axes toform corresponding standing waves aligned in the directions of therespective axes. The plurality of axes includes the vertical axis andone or more axes at angles off the vertical axis. Next, the movablereflecting surface can be moved for sequentially translating thestanding waves, thereby effectively scanning the fluorescent sampledistribution through the standing waves. Such scanning causesfluorophores to emit light within respective pluralities of thinsections of the sample distribution, each plurality of sections beingorthogonal to a respective axis. At least a portion of the light emittedin each section is reflected by the first reflecting surface, and boththe reflected light and the light emitted directly by the respectivesample sections are collected by the objective lens. The direct andreflected light associated with each sample section undergo constructiveand destructive interference to cause fringes in the correspondingemission spectrum. Next, the periodicity and peak wavelengths of therespective emission spectra are spectroscopically analyzed to generatethe image information, which is then tomographically analyzed toreconstruct the three-dimensional structure of the sample distribution.

By utilizing the techniques of spectral self-interference fluorescentmicroscopy, variable standing wave illumination, and rotating apertureinterferometric nanoscopy, three-dimensional imaging of microscopysamples can be achieved with resolution on the order of at least ten totens of nano-meters in one or more dimensions.

Other features, functions, and aspects of the invention will be evidentfrom the Detailed Description of the Invention that follows.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

The invention will be more fully understood with reference to thefollowing Detailed Description of the Invention in conjunction with thedrawings of which:

FIG. 1 is a conceptual representation of structure for performingspectral self-interference fluorescent microscopy according to thepresent invention;

FIG. 2 is a detailed plan view of the structure conceptually representedin FIG. 1;

FIG. 3 is a diagram depicting emission spectra corresponding tomicroscopy samples disposed at respective distances from a reflectingsurface included in the structure of FIG. 2;

FIG. 4 is a plan view of an apparatus for performing three-dimensionaloptical microscopy according to the present invention;

FIG. 5 is a conceptual representation of a technique for coupling alight beam to off-axis angles employed by the apparatus of FIG. 4;

FIG. 6 is a conceptual representation of rotating apertureinterferometric nanoscopy providing sectioning of a microscopy samplealong an off-axis angle employed by the apparatus of FIG. 4;

FIG. 7 is a conceptual representation of vertical sectioning of amicroscopy sample employed by the apparatus of FIG. 4;

FIG. 8 is a conceptual representation of sectioning of a microscopysample along a plurality of axes employed by the apparatus of FIG. 4;and

FIG. 9 is a flow chart depicting a method of performingthree-dimensional optical microscopy according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

U.S. Provisional Patent Application No. 60/256,574 filed Dec. 19, 2000is incorporated herein by reference.

A method and apparatus for performing three-dimensional (3-D) opticalmicroscopy with nano-meter scale resolution is provided. Such highresolution 3-D optical microscopy is achieved by employing spectralself-interference fluorescent microscopy to determine an optical pathlength between at least one fluorescent microscopy sample and areflecting surface, employing variable standing wave illumination toextend the capabilities of the spectral self-interference fluorescentmicroscopy technique to provide vertical sectioning of an arbitrarydistribution of fluorescent samples, and employing rotating apertureinterferometric nanoscopy to provide such sectioning along a pluralityof axes to generate image information suitable for reconstructing thethree-dimensional structure of the sample distribution.

The presently disclosed 3-D optical microscopy apparatus employs thespectral self-interference fluorescent microscopy technique to achieveone-dimensional imaging of at least one microscopy sample withnano-meter scale resolution. FIG. 1 depicts a conceptual representationof a device 200 configured to perform spectral self-interferencefluorescent microscopy, in accordance with the present invention. In theillustrated embodiment, the device 200 includes a housing 201 configuredto house at least one objective lens (not shown) for collecting lightemitted by at least one first fluorescent microscopy sample 220 a. Asshown in FIG. 1, a first portion 214 a of the light emitted by thefluorescent sample 220 a travels directly from the sample 220 a to theobjective lens in the housing 201, while a second portion 214 b of theemission light 214 travels to the housing 201 after being reflected by afirst planar mirror 204. Accordingly, the objective lens in the housing201 collects both the light 214 a emitted directly by the fluorescentsample 220 a and the light 214 b reflected by the first mirror 204.

Those of ordinary skill in the art will appreciate that the emissionlight 214 a emitted directly from the fluorescent sample 220 a to theobjective lens housing 201 and the emission light 214 b reflected by thefirst mirror 204 can undergo constructive and destructive interference.In the illustrated embodiment, the difference between the respectiveoptical path lengths of the direct and reflected light 214 a and 214 bis such that only a relatively small change in wavelength is needed totransition between constructive and destructive interference patterns.The interference between the direct and reflected emission light 214 aand 214 b forms at least one standing wave on the first mirror 204 thatcauses spectral oscillations or “fringes” in the corresponding emissionspectrum.

FIG. 2 depicts an illustrative embodiment of structure configured toimplement the device 200 of FIG. 1. As shown in FIG. 2, the structure200 may be implemented as a Micro Opto Electro Mechanical Systems(MOEMS) device. Specifically, the MOEMS device 200 includes a spacer 202made of, e.g., silicon dioxide (SiO₂) and the first planar mirror 204disposed on a silicon substrate 206 a. The spacer 202 and the firstmirror 204 are configured to form an emission cavity 208. FIG. 2 furthershows the first microscopy sample 220 a disposed at a predetermineddistance d₁ from the first mirror 204 and a second microscopy sample 220b disposed at a predetermined distance d₂ from the mirror 204. Forexample, the microscopy sample 220 b may be immobilized atop a cellularcomponent such as a globular protein and the microscopy sample 220 a maybe immobilized directly on the surface of the spacer 202 usingconventional microscopy sample immobilization techniques.

Because the illustrative spectral self-interference fluorescentmicroscopy technique employs fluorescent probe technology, themicroscopy samples 220 a and 220 b are marked by suitable fluorescentprobes. Further, a light source (not shown) may generate excitationlight (generally shown at reference numeral 216) to illuminate thefluorescent samples 220 a and 220 b, thereby causing the samples 220 aand 220 b to emit light (generally shown at reference numeral 214). Inthe illustrated embodiment, the first mirror 204 is made of a wavelengthselective dielectric configured to reflect the emission wavelengthswhile being transparent to the excitation wavelength. For example, thefirst mirror 204 may comprise SiO₂ and silicon nitride (Si₃N₄). In analternative embodiment, the first mirror 204 may be made of material,e.g., silicon (Si), that reflects both the emission and excitationwavelengths. Moreover, the thickness of the spacer 202 is such that therespective distances d₂ and d₁ of the fluorescent samples 220 a and 220b from the first mirror 204 are several to several tens times thewavelength of the excitation light 216. In the illustrated embodiment,the excitation wavelength ranges from about 500 to 600 nano-meters.

FIG. 3 depicts exemplary emission spectra 300 associated with thefluorescent samples 220 a and 220 b (see FIG. 2). For example, theemission spectrum 302 may be associated with the fluorescent sample 220b immobilized at the predetermined distance d₂ from the first mirror204, and the emission spectrum 304 may be associated with thefluorescent sample 220 a immobilized at the predetermined distance d₁from the first mirror 204 (i.e., on the surface of the spacer 202). Asshown in FIG. 3, both emission spectra 302 and 304 comprise fringescaused by constructive and destructive interference between direct andreflected light emitted by the fluorescent samples 220 a and 220 b,respectively. These spectral fringes impart periodic behavior to theemission spectra 302 and 304, the details of which may be extractedusing conventional mathematical techniques.

It is noted that the periodicity and peak wavelengths of the respectiveemission spectra 302 and 304 are functions of the distances d₂ and d₁between the fluorescent samples 220 a and 220 b and the first mirror204, respectively, and possibly the orientation of the samples 220 a and220 b relative to the mirror 204. In effect, information relating to therespective distances d₂ and d₁ is encoded in the emission spectra 302and 304. It is further noted that the distance information encoded inthe emission spectra 302 and 304 is independent of the fluorescentsample density, the emission intensity, and the excitation fieldstrength. By analyzing the intensity peaks and valleys in the emissionspectra 302 and 304, the location of the fluorescent sample responsiblefor the respective emission signal can be precisely determined, and ahigh level of discrimination between the specifically bound fluorescentsample 220 b and the surface bound fluorescent sample 220 a can beachieved.

For example, differential phase measurements may be performed using theemission spectra 300 to discriminate between the surface bound andspecifically bound microscopy samples 220 a and 220 b. Such a phasedifference is illustrated in a detail view 301 (see FIG. 3) ofrespective intensity peaks in the emission spectra 302 and 304. Bymeasuring the magnitude of this phase difference, the difference (d₂−d₁)between the respective distances from the fluorescent samples 220 a and220 b to the first mirror 204 can be determined with nano-meter scaleresolution.

It is understood that the respective distances between the fluorescentsamples 220 a and 220 b and the first mirror 204 may also be determinedusing direct phase measurements. Moreover, the first mirror 204 may bemoved along an axis passing near or through at least one of the samples220 a and 220 b to cause the pattern of intensity of light in theemission spectra 302 and 304 to shift as a function of wavelength. Forexample, the location of the first mirror 204 may be controlled relativeto the fluorescent samples 220 a and 220 b by a piezo-electric tubescanner.

The presently disclosed spectral self-interference fluorescentmicroscopy technique will be better understood with reference to thefollowing first illustrative example. In this first example, thespectral self-interference microscopy technique is performed by theMOEMS device 200 (see FIG. 2) to determine the respective distances d₂and d₁ from the fluorescent samples 220 a and 220 b to the first mirror204, which is configured to reflect the emission wavelengths and betransparent to the excitation wavelength. For example, the fluorescentsamples 220 a and 220 b may be respective bio specimens comprisingliving tissue.

Specifically, the fluorescent sample 220 b is immobilized at thepredetermined distance d₂ from the first mirror 204 while thefluorescent sample 220 a is immobilized at the predetermined distance d₁from the first mirror 204, i.e., directly on the surface of the spacer202. It is noted that the thickness of the spacer 202 is several toseveral tens times the excitation wavelength. Each fluorescent sample220 a and 220 b emits light in response to being illuminated by theexcitation light, and the emission spectrum associated with eachfluorescent sample 220 a and 220 b is distinguishable by its periodicityand peak wavelengths (i.e., by its frequency and phase).

In this first example, the respective emission spectra associated withthe fluorescent samples 220 a and 220 b add together to form a complexemission spectrum representative of the sum of the individual emissions.Because each emission spectrum is distinguishable by its frequency andphase, conventional Fourier Transform techniques can be used tocalculate the amplitudes and frequencies of the spectra components.Specifically, the complex emission spectrum comprises a plurality ofcomponents, one of which creates the spectral fringe pattern and holdsall of the distance information. As explained above, the fringe patternis given by the interference of the emission light with its reflectionfrom the first mirror 204. The interference component of the intensityfor each emitting sample 220 a and 220 b is given by|1+re^(−ik) ⁰ ^(2(nD+d))|²=1+r ²+2r cos(k ₀2(nD+d)),  (1)in which “r” is the total reflectivity of the first mirror 204, “nD” isthe respective optical path in the spacer 202, and “d” is the distanceof the fluorescent sample from the first mirror 204. It is noted thatthe intensities of the fluorescent samples 220 a and 220 b are addedbecause they are incoherent. Further, each of the fluorescent samples220 a and 220 b makes a unique contribution to the complex emissionspectrum.

The calculated frequency and phase information for the respectiveemission spectra are indicative of the respective distances d₂ and d₁ ofthe samples 220 a and 220 b from the first mirror 204 while thecalculated amplitudes correspond to the fluorescent density at thosedistances. It is noted that such complex emission spectra can besuitably de-convoluted to allow distances between emitting microscopysamples and reflecting surfaces to be determined with a resolution of atleast ten to tens of nano-meters.

The presently disclosed 3-D optical microscopy apparatus employs thevariable standing wave illumination technique to extend the capabilitiesof spectral self-interference microscopy to provide vertical sectioningof an arbitrary distribution of fluorescent samples. To this end, theMOEMS device 200 (see FIG. 2) further includes a movable planar mirror205 disposed on a silicon substrate 206 b and configured to reflect theexcitation light 216. For example, the movable mirror 205 may be abroadband mirror comprising suitable metal and dielectric layers. In theillustrated embodiment, the spacer 202, the first mirror 204 (which istransparent to the excitation light 216), the substrate 206 a, and themovable mirror 205 are configured to form an excitation cavity 210. Itis noted that the excitation cavity 210 comprises an adjustableseparation 212 between the substrate 206 a and the movable mirror 205.For example, the separation 212 in the MOEMS device 200 may be adjustedby controlling the location of the movable mirror 205 relative to thesubstrate 206 a using a piezo-electric tube scanner.

As described above, the excitation light 216 is directed to thefluorescent samples 220 a and 220 b, thereby causing the samples 220 aand 220 b to emit the emission light 214. The excitation light 216 isfurther transmitted through the first mirror 204 to the movable mirror205 along the Z-axis. The direct excitation light 216 and the excitationlight 216 reflected by the movable mirror 205 interfere to form at leastone standing wave 218 on the movable mirror 205 aligned in theZ-direction. By moving the mirror 205 along the Z-axis, the standingwave 218 can be translated for effectively scanning a fluorescent sampledistribution including the fluorescent samples 220 a and 220 b throughthe standing wave 218. It is understood that such translation of thestanding wave 218 may also be performed by varying either the angle ofthe excitation light 216 or the excitation wavelength.

Scanning the distribution of fluorescent samples through the field ofthe standing wave 218 causes fluorophores to emit light within aplurality of thin sections of the sample distribution orthogonal to theZ-axis, as shown in the conceptual representation of FIG. 7. In theillustrated embodiment, the spatial variation of the incident fieldintensity excites the fluorophores within the thin sample sections toattain a resolution of at least ten to tens of nano-meters along theZ-axis.

The presently disclosed variable standing wave illumination techniquewill be better understood with reference to the following secondillustrative example. In this second example, variable standing waveillumination is used in conjunction with the above-described spectralself-interference fluorescent microscopy technique to determine thelocation of at least one broad continuous distribution of fluorescentsamples.

Specifically, a distribution of fluorescent samples such as the samples220 a and 220 b (see FIG. 2) are immobilized at predetermined distances(i.e., at least several to several tens times the excitation wavelength)from the first mirror 204. The fluorescent samples emit light inresponse to being illuminated by the excitation light, and the emissionspectra associated with the respective samples are distinguishable bytheir frequency and phase. Moreover, the excitation light is reflectedby the movable mirror 205 to create at least one standing wave 218aligned in the Z-direction.

In this second example, the movable mirror 205 is moved along the Z-axisto scan the distribution of fluorescent samples through the standingwave 218. In effect, the standing wave 218 is used to obtain thinsections of the sample distribution, thereby discretizing the broaddistribution of fluorophores. Specifically, the distribution of thefluorophores can be represented by profiles A(d₁) . . . A(d_(N)) (seeFIG. 7), which comprise a discrete series of axial slices at distancesd₁ to d_(N) from the first mirror 204. Unknown emission amplitudescorresponding to each slice A(d₁) . . . A(d_(N)) can be determined froma set of spectra measured as the standing wave 218 is scanned. In thisexample, such measured spectra are expressed asS _(j)=Σ_((i=1−N)) I _(j)(d _(i))A _(i) ²[1+R ²+2R cos(2k(nD+d_(i)))],  (2)S _(j)=Σ_((i=1−N)) I _(j)(d _(i))A _(i) ² F _(i),  (3)in which “F_(i)” is an interference term, “I_(j)(d_(i))” is the strengthof the excitation intensity at distance “d_(i)” for standing waveposition “j”, “S_(j)” is the entire observed spectrum, and “A_(I)” isthe emission amplitude from position d_(i). Collecting observations fromN different excitation standing wave field positions yields

$\begin{matrix}{\begin{bmatrix}{S_{1}(k)} \\{S_{2}(k)} \\\vdots \\{S_{N}(k)}\end{bmatrix} = {\begin{bmatrix}{{I_{1}( d_{1} )}F_{1}} & {{I_{1}( d_{2} )}F_{2`}} & \ldots & {{I_{1}( d_{N} )}F_{N}} \\{{I_{2}( d_{1} )}F_{1}} & {{I_{2}( d_{2} )}F_{2}} & \ldots & {{I_{2}( d_{N} )}F_{N}} \\\; & \vdots & \; & \; \\{{I_{N}( d_{1} )}F_{1}} & {{I_{N}( d_{2} )}F_{2}} & \ldots & {{I_{N}( d_{N} )}F_{N}}\end{bmatrix}\begin{bmatrix}A_{1}^{2} \\a_{2}^{2} \\\vdots \\A_{N}^{2}\end{bmatrix}}} & (4)\end{matrix}$which may be solved to recover the fluorophore distribution profilesA(d₁) . . . A(d_(N)).

It is noted that the determination of the vertical distribution of thefluorescent samples can be facilitated by immobilizing one or moresamples at predetermined reference distances (i.e., at least several toseveral tens times the excitation wavelength) from the first mirror 204for use in providing a more complete basis set for subsequentde-convolution of the emission spectra.

FIG. 4 depicts an illustrative embodiment of an apparatus 100 forperforming high resolution 3-D optical microscopy, in accordance withthe present invention. The optical microscopy apparatus 100 includes ahousing 102 configured to house a light source 105 (see FIG. 5) andfirst and second objective lenses 101 and 103 (see FIGS. 5 and 6), aspherical mirror 104, and a plurality of microscope stages 110–112. Thelight source 105 is configured to generate excitation light 108, and theobjective lenses 101 and 103 are configured to direct the excitationlight 108 to a microscopy specimen (“sample”) 114 and collect lightemitted by the sample 114 in response to being illuminated by theexcitation light 108. In the illustrated embodiment, the light source105 is a single mode optical fiber and the excitation light 108comprises laser radiation in the form of a Gaussian beam. Further, eachof the objective lenses 101 and 103 have a relatively high NumericalAperture (e.g., NA≈0.87). The optical microscopy apparatus 100 iscoupleable to a spectrometer 115 configured to generate image data fromspectral information collected via the objective lenses 101 and 103. Forexample, the spectrometer 115 may comprise a grating spectrometer.Further, the spectrometer 115 may provide the image data to a tomographyanalyzer 117 configured to tomographically analyze the image data forreconstructing the three-dimensional structure of the microscopy sample114.

As shown in FIG. 4, the microscopy sample 114 is mounted on at least oneglass cover slide 106, which is directly supported by the XY-stage 111.Further, the spherical mirror 104 is directly supported by the Z-stage112. The XY-stage 111 is configured for adjustably positioning the glasscover slide 106 in the XY-plane to place the microscopy sample 114mounted thereon at the focal point of the excitation light 108, and theZ-stage 112 is configured for adjustably positioning the sphericalmirror 104 in the Z-direction to place the center of the mirror 104 atthe approximate location of the sample 114.

The optical microscopy apparatus 100 (see FIG. 4) employs the rotatingaperture interferometric nanoscopy technique to provide sectioning of anarbitrary distribution of fluorescent samples including the microscopysample 114 along a plurality of axes to generate image informationsuitable for reconstructing the 3-D structure of the sampledistribution. For example, the plurality of axes may include thevertical Z-axis and one or more axes disposed at angles off the verticalaxis.

FIG. 5 depicts the light source 105 and the objective lenses 101 and 103of the optical microscopy apparatus 100 (see FIG. 4) arranged to providethe excitation light 108 at an exemplary off-axis angle. It is notedthat different angles of the excitation light 108 can be attained byvarying the angle of the light source 105 relative to the XY-planeand/or rotating the light source 105, as shown in FIG. 5.

In the illustrated embodiment, the light source 105, (i.e., the singlemode optical fiber) is, in effect, placed at the center of a conjugateimage plane of a 4f microscope confocal system. Further, the objectivelenses 101 and 103 are identical and arranged to reproduce theexcitation light 108 provided by the light source 105 at the location ofthe microscopy sample 114. In this way, the excitation light 108 isoptimally coupled in a single optical mode from the light source 105 tothe microscopy sample 114, and subsequently optimally collected from thesame mode in emission.

Having provided the excitation light 108 to the microscopy sample 114 atthe desired angle, the light 108 is reflected back onto itself by thespherical mirror 104 to create the excitation standing wave 218 (seeFIG. 2). In the illustrated embodiment, the spherical mirror 104 ispositioned by the Z-stage 112 at a distance R (see FIG. 4) from themicroscopy sample 114 that matches spherical phase wave fronts 109 (seeFIG. 6). Specifically, the mirror 104 is configured with a radius thatis greater than the confocal parameter so that the phase wave fronts 109are spherical at the position of the mirror 104. In this configuration,light emitted below the focal plane at all angles by a given fluorophorewithin the microscopy sample 114 is reflected back to that fluorophoresuch that the direct and reflected light travel exactly the same opticalpath.

As a result, the spherical phase wave fronts 109 emitted below the focalplane are reflected back to the point of origin, thereby removing anyangular dependence of the phase difference between the direct andreflected emission light. It is noted that the polarization is alsomapped back onto itself without distortion. It is expected that use ofthe spherical mirror 104 matched to the phase wave fronts 109 and theobjective lenses 101 and 103 having relatively high NA will increase thelateral resolution of the optical microscopy apparatus 100 to at leastten to tens of nano-meters. In contrast, conventional optical microscopyapparatus normally provide diffraction limited lateral resolution, whichis typically on the order of hundreds of nano-meters.

As required in the variable standing wave illumination technique, thespherical mirror 104 is moved to translate the excitation standing wave218 for effectively scanning the distribution of fluorescent samplesalong the vertical axis and at the off-axis angles. As mentioned above,the movement of the spherical mirror 104 may be piezo-electricallycontrolled. Such scanning of the fluorescent sample distribution throughthe field of the standing wave 218 causes fluorophores to emit lightwithin a plurality of thin sample sections orthogonal to the pluralityof axes. This is shown in the conceptual representation of FIG. 8, inwhich the distribution of the fluorophores is represented by at leastone profile A(d_(N)). FIG. 6 depicts another conceptual representationof emitting fluorophores within a plurality of thin sample sections 107at an exemplary off-axis angle. These exemplary fluorophore distributionprofiles comprise image information that can be spectroscopically andtomographically analyzed to reconstruct the three-dimensional structureof the sample distribution. It is noted that the spatial variation ofthe incident field intensity excites the fluorophores within the thinsample sections to attain a resolution of at least ten to tens ofnano-meters along the respective axes. Moreover, the use of thespherical mirror 104 matched to the phase wave fronts and the high NAobjective lenses 101 and 103 increases lateral resolution in planesorthogonal to the respective axes to at least ten to tens ofnano-meters.

The presently disclosed method of performing 3-D optical microscopy isillustrated by reference to FIG. 9. As depicted in step 902, afluorescent microscopy sample is provided that is capable of emittinglight in response to being illuminated by excitation light. Next, amirror is provided, as depicted in step 904, that is reflective at leastat the emission wavelength. The microscopy sample is then immobilized,as depicted in step 906, at a distance from the mirror that is at leastseveral to several tens times the excitation wavelength. Next, theexcitation light is applied, as depicted in step 908, to the microscopysample to induce the sample to fluoresce. An interference pattern isthen provided, as depicted in step 910, representative of theinterference of the sample fluorescence along optical paths directlyfrom the sample and after reflection by the mirror. Finally, theinterference pattern is resolved, as depicted in step 912, to obtain theposition of the microscopy sample with sub-micron resolution.

It will further be appreciated by those of ordinary skill in the artthat modifications to and variations of the above-describedmulti-dimensional optical microscopy technique may be made withoutdeparting from the inventive concepts disclosed herein. Accordingly, theinvention should not be viewed as limited except as by the scope andspirit of the appended claims.

1. Apparatus for determining the position of at least one specimen in anenvironment, comprising: at least one first surface reflective at leastat a wavelength range of interest; said first reflective surface beingdisposed at a predetermined distance from said specimen; said specimenhaving light emission associated therewith; and a spectral analyzerresponsive to light representing an interference of the specimenemission associated with said at least one specimen on paths directlyfrom said at least one specimen and after reflection by said firstreflective surface to provide an interference pattern resolvable forposition of said specimen.
 2. The apparatus of claim 1 wherein saidspectral analyzer includes at least one lens directing radiation fromsaid specimen to said spectral analyzer.
 3. The apparatus of claim 2further including a source of radiation for said specimen to excite saidemission of said specimen.
 4. The apparatus of claim 3 wherein saidsource applies said radiation through at least one lens.
 5. Theapparatus of claim 1 wherein said specimen emissions are characterizedby one or more of spontaneous emission, excitation induced emissions,self emissions of said specimen, and decaying emissions from previouslyexcited emissions.
 6. The apparatus of claim 3 further including asecond reflective surface distant from said first reflective surface,said first reflective surface being disposed between said secondreflective surface and said specimen, said second surface beingreflective at a wavelength of said source and said first surface beingtransmissive at the wavelength of said source.
 7. The apparatus of claim6 wherein: means are provided for moving at least one of said first andsecond reflective surfaces; and said spectrum analyzer includes meansfor moving an angle of view through which said analyzer responds to saidemission and for providing a tomographic representation of theenvironment of said specimen as a function of the angle of view andsurface position.
 8. The apparatus of any one of the previous claimswherein said spectral analyzer comprises a grating spectrometer.
 9. Theapparatus of claim 8 wherein said spectrometer provides a patternrepresenting the intensity of light from said emission as a function ofwavelength.
 10. The apparatus of any claim 9 wherein said firstreflective surface includes an effector operative to move said firstsurface along an axis passing near or through said specimen and said atleast one lens thereby causing the pattern of intensity of light toshift as a function of wavelength.
 11. The apparatus of any claim 1wherein said first reflective surface is spherical with a centersubstantially corresponding to a location of said specimen.
 12. Theapparatus of claim 1 wherein said specimen is a biological specimen andsaid emissions are from a marker associated with said specimen.
 13. Theapparatus of claim 3 wherein said radiation is laser radiation. 14.Apparatus for providing tomographic data representations of at least onespecimen from optical interference patterns, said at least one specimenhaving an optical emission associated therewith, comprising: an opticalsystem for receiving light emission from said specimen over a range ofangles; a first reflective surface responsive to a portion of theemission from said specimen to redirect it toward said optical system toprovide self interference of light in said emission; a radiation source;a second reflective surface responsive to said radiation from saidsource to provide an interference pattern in an environment of saidspecimen, said first reflective surface being disposed between saidsecond reflective surface and said specimen; means for moving the firstand second reflective surfaces to vary the position of said interferencepattern in the environment of said specimen and to vary the interferencepattern of the self interference; and a wavelength analyzer providingsaid tomographic representations as a function of the self interferingemission and the positions of said first and second reflective surfaces.15. The apparatus of claim 14 wherein said first and second surfaces arefocussing.
 16. The apparatus of claim 15 wherein said optical system isconfigured to apply said source radiation to said specimen environmentand to rotate an angle of application and receipt of radiation appliedto said specimen and received from said specimen.
 17. The apparatus ofclaim 16 wherein said optical system includes first and second lensesfor receiving the radiation from the source and directing it to saidspecimen and for receiving emissions from the environment of saidspecimen.
 18. The apparatus of claim 17 wherein at least one of saidfirst and second reflective surfaces are movable in a direction along anaxis which includes said specimen.
 19. A method for determining theposition of at least one specimen in an environment, comprising thesteps of: supporting said specimen in a location a distance from a firstreflective surface; said specimen having light emission associatedtherewith; and spectrally analyzing light representing the interferenceof the specimen emission associated with said specimen on paths directlyfrom said at least one specimen and after reflection by said firstreflective surface to provide an interference pattern resolvable forposition of said specimen.
 20. The method of claim 19 further includingproviding radiation for said specimen to excite said emission of saidspecimen.
 21. The method of claim 19 wherein said specimen emissions arecharacterized by one or more of spontaneous emission, excitation inducedemissions, self emissions of said specimen, and decaying emissions frompreviously excited emissions.
 22. The method of claim 19 furtherincluding the steps of: moving at least one of said first reflectivesurface and a second reflective surface, said first reflective surfacebeing disposed between said second reflective surface and said specimen;moving an angle of view through which said analyzer responds to saidemissions; and providing a tomographic representation of the environmentof said specimen as a function of the angle of view and surfaceposition.
 23. The method of claim 19 further providing a patternrepresenting the intensity of light from said emission as a function ofwavelength.
 24. The method of claim 19 further including moving saidfirst reflective surface along an axis passing near or through saidspecimen thereby causing the pattern of intensity of light to shift as afunction of wavelength.
 25. The method of claim 19 wherein said specimenis a biological specimen and said emission is from a marker associatedwith said specimen.
 26. A method for providing tomographic datarepresentations of specimens from optical interference patterns,comprising the steps of: receiving at an optical system light emissionfrom a specimen over a range of angles; responding to a portion of theemission from said specimen by a first reflective surface to redirectsaid portion toward said optical system to provide self interference oflight in said emission in said optical system; providing with a secondreflective surface an interference pattern in an environment of saidspecimen, said first reflective surface being disposed between saidsecond reflective surface and said specimen; moving at least one of saidfirst and second reflective surfaces to vary the position of saidinterference pattern in the environment of said specimen and to vary theinterference pattern of the self interference; and providing saidtomographic representations as a function of the self interferingemission and the positions of said first and second reflective surfaces.27. The method of claim 26 wherein said optical system is configured toapply said source radiation to said specimen environment and includingthe step of rotating an angle of application and receipt the radiationapplied to said specimen and received from said specimen.
 28. The methodclaim 27 including moving at least one of said first and secondreflective surfaces in a direction along an axis which includes saidspecimen.